Hitoshi Morikawa

Activation of inflammatory signaling by lipopolysaccharide produces a prolonged increase of voluntary alcohol intake in mice.

Previous studies showed that mice with genetic predisposition for high alcohol consumption as well as human alcoholics show changes in brain expression of genes related to immune signaling. In addition, mutant mice lacking genes related to immune function show decreased alcohol consumption (Blednov et al., 2011), suggesting that immune signaling promotes alcohol consumption. To test the possibility that activation of immune signaling will increase alcohol consumption, we treated mice with lipopolysaccaride (LPS; 1mg/kg, i.p.) and tested alcohol consumption in the continuous two-bottle choice test. To take advantage of the long-lasting activation of brain immune signaling by LPS, we measured drinking beginning one week or one month after LPS treatment and continued the studies for several months. LPS produced persistent increases in alcohol consumption in C57BL/6J (B6) inbred mice, FVBxB6F1 and B6xNZBF1 hybrid mice, but not in FVB inbred mice. To determine if this effect of LPS is mediated through binding to TLR4, we tested mice lacking CD14, a key component of TLR4 signaling. These null mutants showed no increase of alcohol intake after treatment with LPS. LPS treatment decreased ethanol-conditioned taste aversion but did not alter ethanol-conditioned place preference (B6xNZBF1 mice). Electrophysiological studies of dopamine neurons in the ventral tegmental area showed that pretreatment of mice with LPS decreased the neuronal firing rate. These results suggest that activation of immune signaling promotes alcohol consumption and alters certain aspects of alcohol reward/aversion.

Recurrent Inhibitory Network among Striatal Cholinergic Interneurons

The striatum plays a central role in sensorimotor learning and action selection. Tonically active cholinergic interneurons in the striatum give rise to dense axonal arborizations and significantly shape striatal output. However, it is not clear how the activity of these neurons is regulated within the striatal microcircuitry. In this study, using rat brain slices, we find that stimulation of intrastriatal cholinergic fibers evokes polysynaptic GABAA IPSCs in cholinergic interneurons. These polysynaptic GABAA IPSCs were abolished by general nicotinic acetylcholine receptor antagonists and also by a specific antagonist of nicotinic receptors containing β2 subunits. Dopamine receptor antagonists or dopamine depletion failed to block polysynaptic IPSCs, indicating that phasic dopamine release does not directly mediate the polysynaptic transmission. Dual recording from pairs of cholinergic interneurons revealed that activation of a single cholinergic interneuron is capable of eliciting polysynaptic GABAA IPSCs both in itself and in nearby cholinergic interneurons. Although polysynaptic transmission arising from a single cholinergic interneuron was depressed during repetitive 2 Hz firing, intrastriatal stimulation reliably evoked large polysynaptic IPSCs by recruiting many cholinergic fibers. We also show that polysynaptic GABAergic inhibition leads to a transient suppression of tonic cholinergic interneuron firing. We propose a novel microcircuit in the striatum, in which cholinergic interneurons are connected to one another through GABAergic interneurons. This may provide a mechanism to convert activation of cholinergic interneurons into widespread recurrent inhibition of these neurons via nicotinic excitation of striatal GABAergic neurons.

Ethanol enhances GABAergic transmission onto dopamine neurons in the ventral tegmental area of the rat.

BACKGROUND Activation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) by ethanol has been implicated in its rewarding and reinforcing effects. At most central synapses, ethanol generally increases inhibitory synaptic transmission; however, no studies have explored the effect of acute ethanol on GABAergic transmission in the VTA. METHODS Whole-cell patch clamp recordings of inhibitory postsynaptic currents (IPSCs) from VTA-DA neurons in midbrain slices from young rats. RESULTS Acute exposure of VTA-DA neurons to ethanol (25 to 50 mM) robustly enhanced GABAergic spontaneous and miniature IPSC frequency while inducing a slight enhancement of spontaneous IPSC (sIPSC) amplitude. Ethanol (50 mM) enhanced paired-pulse depression of evoked IPSCs, further suggesting enhanced GABA release onto VTA-DA neurons. The frequency of sIPSCs was suppressed by the GABA(B) agonist, baclofen (1.25 microM) and enhanced by the antagonist, SCH50911 (20 microM); however, neither appeared to modulate or occlude the effects of ethanol on sIPSC frequency. CONCLUSIONS The present results indicate that ethanol increases postsynaptic GABA(A) receptor sensitivity, enhances action potential-independent GABA release onto VTA-DA neurons, and that this latter effect is independent of GABA(B) auto-receptor inhibition of GABA release.

Dynamic regulation of midbrain dopamine neuron activity: intrinsic, synaptic, and plasticity mechanisms.

Although the roles of dopaminergic signaling in learning and behavior are well established, it is not fully understood how the activity of dopaminergic neurons is dynamically regulated under different conditions in a constantly changing environment. Dopamine neurons must integrate sensory, motor, and cognitive information online to inform the organism to pursue outcomes with the highest reward probability. In this article, we provide an overview of recent advances on the intrinsic, extrinsic (i.e., synaptic), and plasticity mechanisms controlling dopamine neuron activity, mostly focusing on mechanistic studies conducted using ex vivo brain slice preparations. We also hope to highlight some unresolved questions regarding information processing that takes place at dopamine neurons, thereby stimulating further investigations at different levels of analysis.

Social Deprivation Enhances VTA Synaptic Plasticity and Drug-Induced Contextual Learning

Drug addiction is driven, in part, by powerful drug-related memories. Deficits in social life, particularly during adolescence, increase addiction vulnerability. Social isolation in rodents has been used extensively to model the effects of deficient social experience, yet its impact on learning and memory processes underlying addiction remains elusive. Here, we show that social isolation of rats during a critical period of adolescence (postnatal days 21-42) enhances long-term potentiation of NMDA receptor (NMDAR)-mediated glutamatergic transmission in the ventral tegmental area (VTA). This enhancement, which is caused by an increase in metabotropic glutamate receptor-dependent Ca(2+) signaling, cannot be reversed by subsequent resocialization. Notably, memories of amphetamine- and ethanol-paired contextual stimuli are acquired faster and, once acquired, amphetamine-associated contextual memory is more resistant to extinction in socially isolated rats. We propose that NMDAR plasticity in the VTA may represent a neural substrate by which early life deficits in social experience increase addiction vulnerability.

Burst-Timing-Dependent Plasticity of NMDA Receptor-Mediated Transmission in Midbrain Dopamine Neurons

Bursts of spikes triggered by sensory stimuli in midbrain dopamine neurons evoke phasic release of dopamine in target brain areas, driving reward-based reinforcement learning and goal-directed behavior. NMDA-type glutamate receptors (NMDARs) play a critical role in the generation of these bursts. Here we report LTP of NMDAR-mediated excitatory transmission onto dopamine neurons in the substantia nigra. Induction of LTP requires burst-evoked Ca2+ signals amplified by preceding metabotropic neurotransmitter inputs in addition to the activation of NMDARs themselves. PKA activity gates LTP induction by regulating the magnitude of Ca2+ signal amplification. This form of plasticity is associative, input specific, reversible, and depends on the relative timing of synaptic input and postsynaptic bursting in a manner analogous to the timing rule for cue-reward learning paradigms in behaving animals. NMDAR plasticity might thus represent a potential neural substrate for conditioned dopamine neuron burst responses to environmental stimuli acquired during reward-based learning.

ETHANOL ACTION ON DOPAMINERGIC NEURONS IN THE VENTRAL TEGMENTAL AREA: INTERACTION WITH INTRINSIC ION CHANNELS AND NEUROTRANSMITTER INPUTS

The dopaminergic system originating in the midbrain ventral tegmental area (VTA) has been extensively studied over the past decades as a critical neural substrate involved in the development of alcoholism and addiction to other drugs of abuse. Accumulating evidence indicates that ethanol modulates the functional output of this system by directly affecting the firing activity of VTA dopamine neurons, whereas withdrawal from chronic ethanol exposure leads to a reduction in the functional output of these neurons. This chapter will provide an update on the mechanistic investigations of the acute ethanol action on dopamine neuron activity and the neuroadaptations/plasticities in the VTA produced by previous ethanol experience.

GABAergic transmission modulates ethanol excitation of ventral tegmental area dopamine neurons.

Activation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) by ethanol has been implicated in its rewarding and reinforcing effects. We previously demonstrated that ethanol enhances GABA release onto VTA-DA neurons via activation of 5-HT2C receptors and subsequent release of calcium from intracellular stores. Here we demonstrate that excitation of VTA-DA neurons by ethanol is limited by an ethanol-enhancement in GABA release. In this study, we performed whole-cell voltage clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs) and cell-attached recordings of action potential firing from VTA-DA neurons in midbrain slices from young Long Evans rats. Acute exposure to ethanol (75 mM) transiently enhanced the firing rate of VTA-DA neurons as well as the frequency of mIPSCs. Simultaneous blockade of both GABA(A) and GABA(B) receptors (Picrotoxin (75 μM) and SCH50911 (20 μM)) disinhibited VTA-DA firing rate whereas a GABA(A) agonist (muscimol, 1 μM) strongly inhibited firing rate. In the presence of picrotoxin, ethanol enhanced VTA-DA firing rate more than in the absence of picrotoxin. Additionally, a sub-maximal concentration of muscimol together with ethanol inhibited VTA-DA firing rate more than muscimol alone. DAMGO (3 μM) inhibited mIPSC frequency but did not block the ethanol-enhancement in mIPSC frequency. DAMGO (1 and 3 μM) had no effect on VTA-DA firing rate. Naltrexone (60 μM) had no effect on basal or ethanol-enhancement of mIPSC frequency. Additionally, naltrexone (20 and 60 μM) did not block the ethanol-enhancement in VTA-DA firing rate. Overall, the present results indicate that the ethanol enhancement in GABA release onto VTA-DA neurons limits the stimulatory effect of ethanol on VTA-DA neuron activity and may have implications for the rewarding properties of ethanol.

Transcriptional Signatures of Cellular Plasticity in Mice Lacking the α1 Subunit of GABAA Receptors

GABAA receptors mediate the majority of inhibitory neurotransmission in the CNS. Genetic deletion of the α1 subunit of GABAA receptors results in a loss of α1-mediated fast inhibitory currents and a marked reduction in density of GABAA receptors. A grossly normal phenotype of α1-deficient mice suggests the presence of neuronal adaptation to these drastic changes at the GABA synapse. We used cDNA microarrays to identify transcriptional fingerprints of cellular plasticity in response to altered GABAergic inhibition in the cerebral cortex and cerebellum of α1 mutants. In silico analysis of 982 mutation-regulated transcripts highlighted genes and functional groups involved in regulation of neuronal excitability and synaptic transmission, suggesting an adaptive response of the brain to an altered inhibitory tone. Public gene expression databases permitted identification of subsets of transcripts enriched in excitatory and inhibitory neurons as well as some glial cells, providing evidence for cellular plasticity in individual cell types. Additional analysis linked some transcriptional changes to cellular phenotypes observed in the knock-out mice and suggested several genes, such as the early growth response 1 (Egr1), small GTP binding protein Rac1 (Rac1), neurogranin (Nrgn), sodium channel β4 subunit (Scn4b), and potassium voltage-gated Kv4.2 channel (Kcnd2) as cell type-specific markers of neuronal plasticity. Furthermore, transcriptional activation of genes enriched in Bergman glia suggests an active role of these astrocytes in synaptic plasticity. Overall, our results suggest that the loss of α1-mediated fast inhibition produces diverse transcriptional responses that act to regulate neuronal excitability of individual neurons and stabilize neuronal networks, which may account for the lack of severe abnormalities in α1 null mutants.

Previous Ethanol Experience Enhances Synaptic Plasticity of NMDA Receptors in the Ventral Tegmental Area

Alcohol addiction (alcoholism) is one of the most prevalent substance abuse disorders worldwide. Addiction is thought to arise, in part, from a maladaptive learning process in which enduring memories of drug experiences are formed. However, alcohol (ethanol) generally interferes with synaptic plasticity mechanisms in the CNS and thus impairs various types of learning and memory. Therefore, it is unclear how powerful memories associated with alcohol experience are formed during the development of alcoholism. Here, using brain slice electrophysiology in mice, we show that repeated in vivo ethanol exposure (2 g/kg, i.p., three times daily for 7 d) causes increased susceptibility to the induction of long-term potentiation (LTP) of NMDA receptor (NMDAR)-mediated transmission in mesolimbic dopamine neurons, a form of synaptic plasticity that may drive the learning of stimuli associated with rewards, including drugs of abuse. Enhancement of NMDAR plasticity results from an increase in the potency of inositol 1,4,5-trisphosphate (IP3) in producing facilitation of action potential-evoked Ca2+ signals, which is critical for LTP induction. This increase in IP3 effect, which lasts for a week but not a month after ethanol withdrawal, occurs through a protein kinase A (PKA)-dependent mechanism. Corticotropin-releasing factor, a stress-related neuropeptide implicated in alcoholism and other addictions, further amplifies the PKA-mediated increase in IP3 effect in ethanol-treated mice. Finally, we found that ethanol-treated mice display enhanced place conditioning induced by the psychostimulant cocaine. These data suggest that repeated ethanol experience may promote the formation of drug-associated memories by enhancing synaptic plasticity of NMDARs in dopamine neurons.